Hui Wen Chang

Tea polyphenol (−)‐epigallocatechin‐3‐gallate inhibits nicotine‐ and estrogen‐induced α9‐nicotinic acetylcholine receptor upregulation in human breast cancer cells

SCOPE The aim of this research was to explore whether the tea-polyphenol (-)-epigallocatechin-3-gallate (EGCG) could be used as a potential agent for blocking smoking (nicotine, Nic)- or hormone (estradiol, E2)-induced breast cancer cell proliferation through inhibition of a common signaling pathway. METHODS AND RESULTS To explore whether Nic (>0.1 μM, 24 h) and E2 (>1 nM, 24 h) significantly increased α9-nicotinic acetylcholine (α9-nicotinic acetylcholine receptor (nAChR)) mRNA and protein expression levels, real-time PCR and immunoblotting analysis experiments were performed in human breast cancer (MCF-7) cells. Luciferase promoter activity experiment was performed to test the α9-nAChR promoter activity affected by Nic, E2 or EGCG. The results indicate that treatment with EGCG (1 μM) profoundly decreases Nic- and E2-induced MCF-7 proliferation by down regulating α9-nAChR expression. The α9-nAChR promoter activity is significantly induced by 24-h treatment with Nic (10 μM) or E2 (10 nM) (>1.8 and ∼2.3-fold, respectively) in MCF-7 cells. Pretreatment with EGCG eliminated the Nic- and E2-induced α9-nAChR promoter-dependent luciferase activity. We further demonstrate that combined treatment with EGCG profoundly inhibits [3H]-Nic/ α9-nAChR binding activity in breast cancer cells. CONCLUSIONS We found that the EGCG could be used as an agent for blocking smoking (Nic)- or hormone (E2)-induced breast cancer cell proliferation by inhibiting of α9-nAChR signaling pathway. This study reveals the novel antitumor mechanisms of EGCG, and these results may have significant applications for chemopreventive purposes in human breast cancer.

Combination Treatment with Luteolin and Quercetin Enhances Antiproliferative Effects in Nicotine-Treated MDA-MB-231 Cells by Down-regulating Nicotinic Acetylcholine Receptors

Large-scale epidemiological cohort studies performed in the United States indicate that breast cancer risk is associated with active and passive smoking. As of yet, however, there is no direct evidence of antitumor effects by agents that block the effect of tobacco compound nicotine (Nic) on relevant nicotinic receptors (nAChR) involved in breast tumorigenesis. In the present study, the expression profiles of different nAChR subunits in the human breast cancer cell line (MDA-MB-231) were characterized by RT-PCR. Nic (>0.1 microM, 6 h) significantly increased alpha9-nAChR mRNA and protein expression levels in human breast cancer cells (MDA-MB-231 cells). On the other hand, combined treatment with luteolin (Lut, 0.5 microM) and quercetin (Que, 0.5 microM) profoundly decreased MDA-MB-231 proliferation by down-regulating alpha9-nAChR expression. MDA-MB-231 cells were cultured in soft agar to evaluate anchorage-independent colony formation; combined treatment of Lut+Que inhibited Nic-induced MDA-MB-231 colony formation. Interestingly, the number of colonies formed was profoundly reduced in alpha9-nAChR knockdown (Si alpha9) cells in the combined (Lut+Que)-treated group as compared to the relevant control groups. Such results show that Lut- or Que-induced antitransforming activities were not limited to specific inhibition of the alpha9-nAChR receptor. Both alpha5- and alpha9-nAChR appear to be important molecular targets for Lut- and Que-induced antitumor effects in human breast cancer cells.

In vivo antitumor effects of 4,7-dimethoxy-5-methyl-1,3-benzodioxole isolated from the fruiting body of Antrodia camphorata through activation of the p53-mediated p27/Kip1 signaling pathway.

In this study, 4,7-dimethoxy-5-methyl-1,3-benzodioxole (SY-1) was isolated from three different sources of dried Antrodia camphorata (AC) fruiting bodies. AC is a medicinal mushroom that grows on the inner heartwood wall of Cinnamomum kanehirai Hay (Lauraceae), which is an endemic species that is used in Chinese medicine for its antitumor properties. We demonstrated that SY-1 [given as a 1-30 mg/kg body weight intraperitoneal (ip) injection three times per week] profoundly decreased the growth of COLO-205 human colon cancer cell tumor xenografts in an athymic nude mouse model. We further demonstrated that significant AC extract-mediated antitumor effects were observed at the highest concentration (5 g/kg body weight/day). No gross toxicity signs were observed (i.e., body weight changes, general appearance, or individual organ effects). Frozen COLO-205 xenograft tumors were pulverized in liquid N(2), and the expression of cell cycle regulatory proteins was detected by immunoblotting. We found that the p53-mediated p27/Kip1 protein was significantly induced in the low-dose (1 mg/kg body weight) SY-1-treated tumors, whereas the p21/Cip1 protein levels did not change. The G0/G1 phase cell cycle regulators induced by SY-1 were also associated with a significant decrease in cyclins D1, D3, and A. These results provide further evidence that SY-1 may have significance for cancer chemotherapy.

Luteolin sensitises drug-resistant human breast cancer cells to tamoxifen via the inhibition of cyclin E2 expression

Luteolin is a flavonoid that has been identified in many plant tissues and exhibits chemopreventive or chemosensitising properties against human breast cancer. However, the oncogenic molecules in human breast cancer cells that are inhibited by luteolin treatment have not been identified. This study found that the level of cyclin E2 (CCNE2) mRNA was higher in tumour cells (4.89-fold, (∗)P=0.005) than in normal paired tissue samples as assessed using real-time reverse-transcriptase polymerase chain reaction (RT-PCR) analysis (n=257). Further, relatively high levels of CCNE2 protein expression were detected in tamoxifen-resistant (TAM-R) MCF-7 cells. These results showed that the level of CCNE2 protein expression was specifically inhibited in luteolin-treated (5μM) TAM-R cells, either in the presence or absence of 4-OH-TAM (100nM). Combined treatment with 4-OH-TAM and luteolin synergistically sensitised the TAM-R cells to 4-OH-TAM. The results of this study suggest that luteolin can be used as a chemosensitiser to target the expression level of CCNE2 and that it could be a novel strategy to overcome TAM resistance in breast cancer patients.

Long-Term Ethanol Exposure-Induced Hepatocellular Carcinoma Cell Migration and Invasion through Lysyl Oxidase Activation Are Attenuated by Combined Treatment with Pterostilbene and Curcumin Analogues

Ethanol consumption induces hepatocellular carcinoma (HCC) cell metastasis by changing the extracellular matrix (ECM). Lysyl oxidase (LOX) catalyzes the cross-linkage of collagen or elastin in the ECM. LOX protein and mRNA overexpression (>21-fold compared with controls, n = 6) was detected in cirrhotic HCC patients with a history of alcoholism. LOX protein expression was induced in HCC cells after long-term treatment with ethanol (10 mM) for 20-40 passages (denoted E20-E40 cells). Pterostilbene (PSB, 1 μM) displayed significant potency to reduce LOX-mediated activity in E40 cells when combined with curcumin and its analogues. The ability of E40 cells to form colonies in soft agar was reduced by both genetic depletion of LOX and by chemical inhibitors of LOX expression. This study suggests that targeting LOX expression with food components such as PSB and curcumin may be a novel strategy to overcome ethanol-induced HCC cell metastasis in liver cancer patients.

The in vivo antitumor effects on human COLO 205 cancer cells of the 4,7-dimethoxy-5-(2-propen-1-yl)-1,3-benzodioxole (apiole) derivative of 5-substituted 4,7-dimethoxy-5-methyl-l,3-benzodioxole (SY-1) isolated from the fruiting body of Antrodia camphorate.

CONTEXT The compound 4,7-dimethoxy-5-(2-propen-1-yl)-1,3-benzodioxole (apiole) has been isolated from several different plant species, including Petroselinum sativum. Our recent study found that apiole is a chemical derivative of 4,7-dimethoxy-5-methyl-l,3-benzodioxole (SY-1), which has been isolated from dried Antrodia camphorata (AC ) fruiting bodies, a traditional Chinese medicine with antitumor properties. AIMS Our previous in vitro study demonstrated that apiole inhibits the growth of human colon (COLO 205) cancer cells through the arrest of the cell cycle in G0/G1 phase. The in vivo antitumor effects of apiole were evaluated in this study. SETTING AND DESIGN Apiole was administered to mice at 1-30 mg/kg body weight through intraperitoneal (I.P.) injection three times per week (defined as a dosage of 1×-30×). MATERIALS AND METHODS The in vivo antitumor effects of apiole were evaluated in mice with xenografts of COLO 205 cells. STATISTICAL ANALYSIS All of the data are reported as the means ± S.E. Comparisons were performed with a one-way analysis of variance (ANOVA) followed by a Fishers least significant difference test. Significance was defined as P < 0.05. RESULTS Apiole (> 1×) markedly decreased the growth of COLO 205 human colon cancer cell tumor xenografts in an athymic nude mouse model system through the up-regulation of cell cycle regulators, such as p53, p21/Cip1, and p27/Kip1. The apiole-induced increase in G0/G1 phase cell cycle regulators was also associated with a significant decrease in the expression of cyclins D1 and D3. Surprisingly, statistically significantly higher tumor volumes were observed in mice that received 5× apiole compared with 30× apiole-treated mice (P < 0.05). No gross signs of toxicity were observed (e.g., body weight changes, general appearance, or individual organ effects) in any group. CONCLUSIONS Our results show, for the first time, the promising antitumor effects of apiole against colon tumors in an in vivo xenograft model.

Protein phosphatase Mg2+/Mn2+ dependent 1F promotes smoking-induced breast cancer by inactivating phosphorylated-p53-induced signals

We previously demonstrated that the activation of α9-nicotinic acetylcholine receptor (α9-nAchR) signaling by smoking promotes breast cancer formation. To investigate the downstream signaling molecules involved in α9-nAChR-induced breast tumorigenesis, we used real-time polymerase chain reactions and Western blotting to assess expression of protein phosphatase Mg2+/Mn2+ dependent 1F (PPM1F), a Ser/Thr protein phosphatase, in human breast cancer samples (n=167). Additionally, stable PPM1F-knockdown and -overexpressing cell lines were established to evaluate the function of PPM1F. The phosphatase activity of PPM1F in nicotine-treated cells was assessed through Western blotting, confocal microscopy, and fluorescence resonance energy transfer. Higher levels of PPM1F were detected in the breast cancer tissues of heavy smokers (n=7, 12.8-fold) greater than of non-smokers (n= 28, 6.3-fold) (**p=0.01). In vitro, nicotine induced PPM1F expression, whereas α9-nAChR knockdown reduced the protein expression of PPM1F. A series of biochemical experiments using nicotine-treated cells suggested that the dephosphorylation of p53 (Ser-20) and BAX (Ser-184) by PPM1F is a critical posttranslational modification, as observed in breast cancer patients who were heavy smokers. These observations indicate that PPM1F may be a mediator downstream of α9-nAChR that activates smoking-induced carcinogenic signals. Thus, PPM1F expression could be used for prognostic diagnosis or inhibited for cancer prevention and therapy.

Long-term ethanol exposure causes human liver cancer cells to become resistant to mitomycin C treatment through the inactivation of bad-mediated apoptosis

The aim of this study was to test whether long‐term ethanol consumption confers therapeutic resistance to human liver cancer patients infected with hepatitis B virus (HBV). Chronic ethanol‐treated cells were established by consecutively culturing a human hepatocellular carcinoma cell line, Hep 3B, which contains integrated HBV sequences, for 20–40 passages with or without 10 mM ethanol (designated as E20–E40 and C20–C40, respectively). Flow cytometry analysis demonstrated that a growth promoting effect of long‐term ethanol treatment was induced in the E40 cells through preferential acceleration of S‐phase in these cells. Lower protein expression levels of p16, p21/Cip1, and p27/Kip1 were detected in the ethanol‐treated E40 cells. We further demonstrated that long‐term ethanol‐treated E40 cells develop drug resistance in response to mitomycin C (MMC) treatment (>8 µM). Immunoblot analysis revealed that caspase‐8‐mediated mitochondrial apoptotic signals (such as Bad) were inactivated in the MMC‐resistant E40 cells. Immunoprecipitation experiments demonstrated that the sequestration of phosphorylated Bad (Ser‐112) through its binding with 14‐3‐3 was detected more profoundly in the MMC‐resistant E40 cells. Next, we examined the therapeutic efficacy of MMC (10 mg MMC/kg body weight, three times per week) in severe combined immunodeficient (SCID) mice bearing E40‐ and C40‐xenografted tumors. Significant reductions (>3‐fold) in tumor growth were detected in MMC‐treated C40‐xenografted mice. In vivo and in vitro studies demonstrated that AKT‐ and extracellular signal‐regulated kinase (ERK)‐mediated survival factors inhibited the Bad‐induced mitochondrial apoptotic signals that were involved in E40 tumor cells and that conferred resistance to MMC.

DNA primase polypeptide 1 (PRIM1) involves in estrogen-induced breast cancer formation through activation of the G2/M cell cycle checkpoint: DNA primase polypeptide 1 (PRIM1) involved in estrogen-induced breast cancer formation

The DNA primase polypeptide 1 (PRIM1) is responsible for synthesizing small RNA primers for Okazaki fragments generated during discontinuous DNA replication. PRIM1 mRNA expression levels in breast tumor samples were detected by real‐time PCR analysis. Xenografted tumor model was established to study the carcinogenic role of PRIM1 and its potential therapeutic applications. The average PRIM1 mRNA (copy number × 103/μg) expression was 4.7‐fold higher in tumors than in normal tissue (*p = 0.005, n = 254). PRIM1 was detected preferentially at a higher level (>40‐fold) in poorly differentiated tumor tissues (n = 46) compared with more highly differentiated tumors tissues (n = 10) (*p = 0.005). Poor overall survival rate was correlated to the estrogen receptor positive (ER+, n = 20) patients with higher PRIM1 expression when compare to the ER− (n = 10) patients (Chi Square test, p = 0.03). Stable expression of PRIM1‐siRNA in the ER+ BT‐474 cells‐xenograft tumors significantly reduced tumor volume in SCID mice (*p = 0.005). The anti‐tumoral effects of inotilone isolated from Phellinus linteus was tested and had significant effects on the inhibition of PRIM1 protein expression in ER+ breast cancer cells. In vivo study was performed by administering inotilone (10 mg/kg, twice a week for 6 weeks), which resulted in significantly reduced BT‐474‐xenografted tumor growth volume compared with control (n =5 per group, *p < 0.05). This study provides evidences for the prognostic effects of PRIM1 with poor overall survival rate in the ER+ patients and will be valuable to test for therapeutic purpose.

The impact of the effectiveness of GATA3 as a prognostic factor in breast cancer

The transcription factor GATA3 plays a significant role in mammary gland development and differentiation. We analyzed expression of GATA3 in breast cancer (BC) cell lines and clinical specimens from BC patients in Taiwan. Semiquantitative reverse-transcription polymerase chain reaction (RT-PCR), quantitative real-time PCR were carried out to determine the mRNA level of GATA3 from 241 pairs of matched tumor and adjacent normal tissues from anonymous female donors. GATA3 immunohistochemistry (IHC) staining and H-score were performed (n = 25). Inducing and silencing of GATA3 were done by exposure MCF-7 cell line to nicotine or curcumin, respectively. GATA3 expression was detected in most of the estrogen receptor-positive (ER+) tumor specimens (176/241, 73%) compared with paired normal tissues (65/241, 27%) (P < .001). The GATA3 level was highest in Luminal A, and independent t-tests revealed higher GATA3 was associated with ER+ (P = .018) and BC stages (stage II, and stage IV). Nuclear protein expression of GATA3 was detected in tumor tissues (P < .001) with higher H-score in Luminal A patients (P = .012). Kaplan-Meier survival analyses showed that ER+/progesterone receptor (PgR)+ and lower grade BC patients with relatively high GATA3 had better clinical overall survival (OS). GATA3 regulates ERα and BCL-2 as BC luminal subtype markers. Cox univariate and multivariate analyses demonstrated that the expression of GATA3 was an effective predictor of the risk of death. We demonstrated a correlation between GATA3 expression and only ER+ and suggest that a higher GATA3 expression is a good prognostic factor for OS for ER+ BC patients.